(2S,3R,4S,5S,6R)-6-(hydroxymethyl)oxane-2,3,4,5-tetrol

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A maleic acid ester of an APG was prepared as follows. To a 1 L erlenmeyer flask, containing a magnetic stir bar, was added 185.1 g anhydrous n-butanol (Aldrich, 99.8%), 36.1 g n-octanol (Aldrich, 99+%), and 2.0 g deionized water. To the stirred mixture, 0.184 g (100 mL) of concentrated sulfuric acid (J. T. Baker, 96.6%) was added using a 1 mL glass syringe. This mixture was added to a 500 mL three necked round bottom flask containing 50.0 g of anhydrous α-D-glucose (Aldrich, 96%) and a concave magnetic stir bar. The flask was fitted with a thermocouple probe, a dry air intake, and a 25 mL Barrett receiver on which two glass condensers were mounted, which were connected to a gas bubbler. The condensate collection side of the Barrett receiver was filled with n-heptane, and the gas flow-through side was wrapped in cotton wool for the purpose of insulation. Dry air, passed over a 10 inch column filled with dry molecular sieves and Drierite, was passed through the liquid phase in the round bottom flask. The flask was heated for 4 hours at about 95° to 100° C. using a temperature controlled oil bath. Approximately 12 mL of condensate water was collected in the Barrett receiver as a result of glucose oligomerization reaction and the aldol condensation reaction to give alkylation at the C1 position. The white suspension of sugar particles disappeared as the reaction from glucose to APG proceeded until a clear solution was obtained. This demonstrated that the APG is soluble in the alcohol. The resulting APG solution was colorless, indicating that byproduct formation of colored bodies, such as furfurals, was minimized. To a 1 L erlenmeyer flask, containing a magnetic stir bar The flask was fitted with a thermocouple probe The condensate collection side of the Barrett receiver was filled with n-heptane Dry air filled with dry molecular sieves and Drierite The flask was heated for 4 hours at about 95° to 100° C. Approximately 12 mL of condensate water was collected in the Barrett receiver as

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of α-D-glucose (4.5 g) in water (10 ml), a solution of cupric acetate (20 g) in methanol (250 ml) was added, and the resultant mixture was heated on a water bath for 1 hour. The reaction mixture was cooled, and the precipitated cuprous oxide was eliminated by filtration. Hydrogen sulfide gas was introduced into the filtrate for about 1 minute to precipitate the unreacted cupric acetate in the form of cupric sulfide. After elimination of the precipitate by filtration, the filtrate was treated with a small amount of activated charcoal and concentrated under reduced pressure to give glucosone in a syrupy state. The reaction mixture was cooled the precipitated cuprous oxide was eliminated by filtration Hydrogen sulfide gas was introduced into the filtrate for about 1 minute to precipitate the unreacted cupric acetate in the form of cupric sulfide After elimination of the precipitate by filtration the filtrate was treated with a small amount of activated charcoal concentrated under reduced pressure

Conditions: temp:97.51; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A maleic acid ester of an APG was prepared as follows. To a 1 L erlenmeyer flask, containing a magnetic stir bar, was added 185.1 g anhydrous n-butanol (Aldrich, 99.8%), 36.1 g n-octanol (Aldrich, 99+%), and 2.0 g deionized water. To the stirred mixture, 0.184 g (100 mL) of concentrated sulfuric acid (J. T. Baker, 96.6%) was added using a 1 mL glass syringe. This mixture was added to a 500 mL three necked round bottom flask containing 50.0 g of anhydrous α-D-glucose (Aldrich, 96%) and a concave magnetic stir bar. The flask was fitted with a thermocouple probe, a dry air intake, and a 25 mL Barrett receiver on which two glass condensers were mounted, which were connected to a gas bubbler. The condensate collection side of the Barrett receiver was filled with n-heptane, and the gas flow-through side was wrapped in cotton wool for the purpose of insulation. Dry air, passed over a 10 inch column filled with dry molecular sieves and Drierite, was passed through the liquid phase in the round bottom flask. The flask was heated for 4 hours at about 95 to 100° C. using a temperature controlled oil bath. Approximately 12 mL of condensate water was collected in the Barrett receiver as a result of glucose oligomerization reaction and the aldol condensation reaction to give alkylation at the C1 position. The white suspension of sugar particles disappeared as the reaction from glucose to APG proceeded until a clear solution was obtained. This demonstrated that the APG is soluble in the alcohol. The resulting APG solution was colorless, indicating that byproduct formation of colored bodies, such as furfurals, was minimized. To a 1 L erlenmeyer flask, containing a magnetic stir bar The flask was fitted with a thermocouple probe The condensate collection side of the Barrett receiver was filled with n-heptane Dry air filled with dry molecular sieves and Drierite Approximately 12 mL of condensate water was collected in the Barrett receiver as

Conditions: temp:97.51; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A maleic acid ester of an APG was prepared as follows. To a 1 L erlenmeyer flask, containing a magnetic stir bar, was added 185.1 g anhydrous n-butanol (Aldrich, 99.8%), 36.1 g n-octanol (Aldrich, 99+%), and 2.0 g deionized water. To the stirred mixture, 0.184 g (100 mL) of concentrated sulfuric acid (J. T. Baker, 96.6%) was added using a 1 mL glass syringe. This mixture was added to a 500 mL three necked round bottom flask containing 50.0 g of anhydrous α-D-glucose (Aldrich, 96%) and a concave magnetic stir bar. The flask was fitted with a thermocouple probe, a dry air intake, and a 25 mL Barrett receiver on which two glass condensers were mounted, which were connected to a gas bubbler. The condensate collection side of the Barrett receiver was filled with n-heptane, and the gas flow-through side was wrapped in cotton wool for the purpose of insulation. Dry air, passed over a 10 inch column filled with dry molecular sieves and Drierite, was passed through the liquid phase in the round bottom flask. The flask was heated for 4 hours at about 95 to 100° C. using a temperature controlled oil bath. Approximately 12 mL of condensate water was collected in the Barrett receiver as a result of glucose oligomerization reaction and the aldol condensation reaction to give alkylation at the C1 position. The white suspension of sugar particles disappeared as the reaction from glucose to APG proceeded until a clear solution was obtained. This demonstrated that the APG is soluble in the alcohol. The resulting APG solution was colorless, indicating that byproduct formation of colored bodies, such as furfurals, was minimized. To a 1 L erlenmeyer flask, containing a magnetic stir bar The flask was fitted with a thermocouple probe The condensate collection side of the Barrett receiver was filled with n-heptane Dry air filled with dry molecular sieves and Drierite Approximately 12 mL of condensate water was collected in the Barrett receiver as

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Sulfuric acid (40 ml) is added dropwise to a solution of α-D-glucose (41) (50 g, 0,28 mol) in acetone (1 l) at 0° C. The reaction mixture is stirred for 24 h and then it is neutralized using sodium hydroxide (6 M). The acetone is evaporated and the aqueous layer is extracted with chloroform (X2). The chloroform is dried (MgSO4) filtered and evaporated. Crystallization from cyclohexane yielded the di-isopropylidene glucose (42) (41 g, 57%). The acetone is evaporated the aqueous layer is extracted with chloroform (X2) The chloroform is dried (MgSO4) filtered evaporated Crystallization from cyclohexane

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: AmB (800 mg, 0.86 mmol) was dissolved in 16 mL anhydrous dimethylformamide (DMF) and stirred for 5 min. α-D-Glucose (316 mg, 1.72 mmol) was added. The resulting solution was sealed to the atmosphere and placed in a water bath equilibrated to 37° C. for 6 hr. The reaction was followed by LCMS until judged complete. The reaction product was used directly in the amidation reaction, described infra, without purification. The corresponding D-galactose, D-maltose, D-cellobiose and α-D-galactose glycosylated rearrangement products were obtained using the same procedure, but substituting D-galactose (315 mg, 1.72 mmol), D-maltose hydrate (620 mg, 1.72 mmol), D-cellobiose (588 mg, 1.72 mmol) and α-D-lactose hydrate (620 mg, 1.72 mmol), respectively, for α-D-glucose. was added The resulting solution was sealed to the atmosphere The reaction product was used directly in the amidation reaction without purification

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: AmB (800 mg, 0.86 mmol) was dissolved in 16 mL anhydrous dimethylformamide (DMF) and stirred for 5 min. α-D-Glucose (316 mg, 1.72 mmol) was added. The resulting solution was sealed to the atmosphere and placed in a water bath equilibrated to 37° C. for 6 hr. The reaction was followed by LCMS until judged complete. The reaction product was used directly in the amidation reaction, described infra, without purification. The corresponding D-galactose, D-maltose, D-cellobiose and α-D-galactose glycosylated rearrangement products were obtained using the same procedure, but substituting D-galactose (315 mg, 1.72 mmol), D-maltose hydrate (620 mg, 1.72 mmol), D-cellobiose (588 mg, 1.72 mmol) and α-D-lactose hydrate (620 mg, 1.72 mmol), respectively, for α-D-glucose. was added The resulting solution was sealed to the atmosphere The reaction product was used directly in the amidation reaction without purification

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A maleic acid ester of an APG was prepared as follows. To a 1 L erlenmeyer flask, containing a magnetic stir bar, was added 185.1 g anhydrous n-butanol (Aldrich, 99.8%), 36.1 g n-octanol (Aldrich, 99+%), and 2.0 g deionized water. To the stirred mixture, 0.184 g (100 mL) of concentrated sulfuric acid (J. T. Baker, 96.6%) was added using a 1 mL glass syringe. This mixture was added to a 500 mL three necked round bottom flask containing 50.0 g of anhydrous α-D-glucose (Aldrich, 96%) and a concave magnetic stir bar. The flask was fitted with a thermocouple probe, a dry air intake, and a 25 mL Barrett receiver on which two glass condensers were mounted, which were connected to a gas bubbler. The condensate collection side of the Barrett receiver was filled with n-heptane, and the gas flow-through side was wrapped in cotton wool for the purpose of insulation. Dry air, passed over a 10 inch column filled with dry molecular sieves and Drierite, was passed through the liquid phase in the round bottom flask. The flask was heated for 4 hours at about 95° to 100° C. using a temperature controlled oil bath. Approximately 12 mL of condensate water was collected in the Barrett receiver as a result of glucose oligomerization reaction and the aldol condensation reaction to give alkylation at the C1 position. The white suspension of sugar particles disappeared as the reaction from glucose to APG proceeded until a clear solution was obtained. This demonstrated that the APG is soluble in the alcohol. The resulting APG solution was colorless, indicating that byproduct formation of colored bodies, such as furfurals, was minimized. To a 1 L erlenmeyer flask, containing a magnetic stir bar The flask was fitted with a thermocouple probe The condensate collection side of the Barrett receiver was filled with n-heptane Dry air filled with dry molecular sieves and Drierite The flask was heated for 4 hours at about 95° to 100° C. Approximately 12 mL of condensate water was collected in the Barrett receiver as

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of α-D-glucose (4.5 g) in water (10 ml), a solution of cupric acetate (20 g) in methanol (250 ml) was added, and the resultant mixture was heated on a water bath for 1 hour. The reaction mixture was cooled, and the precipitated cuprous oxide was eliminated by filtration. Hydrogen sulfide gas was introduced into the filtrate for about 1 minute to precipitate the unreacted cupric acetate in the form of cupric sulfide. After elimination of the precipitate by filtration, the filtrate was treated with a small amount of activated charcoal and concentrated under reduced pressure to give glucosone in a syrupy state. The reaction mixture was cooled the precipitated cuprous oxide was eliminated by filtration Hydrogen sulfide gas was introduced into the filtrate for about 1 minute to precipitate the unreacted cupric acetate in the form of cupric sulfide After elimination of the precipitate by filtration the filtrate was treated with a small amount of activated charcoal concentrated under reduced pressure

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of α-D-glucose (4.5 g) in water (10 ml), a solution of cupric acetate (20 g) in methanol (250 ml) was added, and the resultant mixture was heated on a water bath for 1 hour. The reaction mixture was cooled, and the precipitated cuprous oxide was eliminated by filtration. Hydrogen sulfide gas was introduced into the filtrate for about 1 minute to precipitate the unreacted cupric acetate in the form of cupric sulfide. After elimination of the precipitate by filtration, the filtrate was treated with a small amount of activated charcoal and concentrated under reduced pressure to give glucosone in a syrupy state. The reaction mixture was cooled the precipitated cuprous oxide was eliminated by filtration Hydrogen sulfide gas was introduced into the filtrate for about 1 minute to precipitate the unreacted cupric acetate in the form of cupric sulfide After elimination of the precipitate by filtration the filtrate was treated with a small amount of activated charcoal concentrated under reduced pressure

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Rebaudioside C, 3-sulfopropyl ester, potassium salt prepared as in Part B. was incubated anaerobically for three days at 37° C. with 5% fresh rat cecal contents at concentrations of 0.25, 0.5, and 1.0 mg/ml, in sterile Krebs-Ringer 0.25 M phosphate buffer (pH 7.4) containing 0.25 mg/ml dithiothreitol and 0.25 mg/ml α-D-glucose. TLC [silica gel F-254; CHCl3 :CH3OH:H2O (15:10:2)] and HPLC [30 cm C-18 on μ-Bondapak; 15 min linear gradient of 10-40% CH3CN in 0.005 M KH2PO4 (pH 3.45); 200 nm] analysis showed all the rebaudioside C, 4-sulfopropyl ester (Rf =0.42; tR =14.0 min) to have been consumed within 24 h to yield apparently only the sulfopropyl ester of steviol, (Rf =0.63; tR =16.3 min). No steviol (Rf =0.95; tR =31.3 min) was detected. After 3 days the bacterial cells were sedimented by centrifugation. The sediment was extracted (THF), as was the lyophilized supernatant. HPLC analysis for steviol of the two THF extracts showed none to be detectable. With a detection limit of 0.05 μg, as little as 0.03 and 0.13 percent degradation to steviol could have been detected for the sediment and supernatant fractions, respectively. to have been consumed within 24 h

Conditions: temp:34.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Alkaloid effects on [3H]overflow from rat striatal slices preloaded with [3H]DA and hippocampal slices preloaded with [3H]NE were determined using modifications of a previously published method (Dwoskin and Zahniser, 1986). Briefly, coronal striatal or hippocampal slices (500 μm, 6-8 mg) were incubated in Krebs' buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 1.0 mM NaH2PO4, 1.3 mM CaCl2, 11.1 mM α-D-glucose, 25 mM NaHCO3, 0.11 mM L-ascorbic acid and 4.0 mM disodium ethylenediaminetetraacetate; pH 7.4, and saturated with 95% O2/5% CO2) in a metabolic shaker at 34° C. for 30 min. Striatal or hippocampal slices were incubated in fresh buffer (6-8 slices/3 mL) containing 0.1 μM [3H]DA (3,4-[7-3H]-dihydroxyphenylethylamine; specific activity 28 Ci/mMol) or 0.1 μM [3H]NE (levo-[7-3H]-norepinephrine; specific activity 14.4 Ci/mMol), respectively, for an additional 30 min. After rinsing, each slice was transferred to a glass superfusion chamber maintained at 34° C. and was superfused at 1 mL/min with oxygenated Krebs' buffer containing pargyline (10 μM) and nomifensine (10 μL) or pargyline and desipramine (10 μL) in experiments assessing [3H]DA and [3H]NE overflow, respectively. After 60 min of superfusion, three 5-min samples (5 mL) were collected to determine basal [3H]outflow. After collection of the third basal sample, slices from an individual rat were superfused in the absence or presence of a single concentration of analog, which remained in the buffer until the end of the experiment. Each slice was exposed to only one concentration of analog. After 30 min, S(−)-nicotine (10 μM) was added to the buffer containing analog, and superfusion continued for an additional 60 min. These experiments utilized a repeated measures design, such that the analog concentration-effect was determined in both the absence and presence of S(−)-nicotine using striatal or hippocampal slices from a single rat. Additionally, one striatal or hippocampal slice was superfused in the absence of analog and constituted the control condition. At the end of the experiment, each slice was solubilized with TS-2. The pH and volume of the solubilized tissue samples were adjusted to those of the superfusate samples. Radioactivity in the superfusate and tissue samples was determined by liquid scintillation spectroscopy (Packard model B1600 TR Scintillation Counter, Downer's Grove, Ill.). After rinsing buffer containing pargyline (10 μM) and nomifensine (10 μL) or pargyline and desipramine (10 μL) in experiments After 60 min of superfusion, three 5-min samples (5 mL) were collected After collection of the third basal sample, slices from an individual rat were superfused in the absence or presence of a single concentration of analog, which After 30 min S(−)-nicotine (10 μM) was added to the buffer containing analog, and superfusion continued for an additional 60 min such that the analog concentration-effect